Mining the deep Red-Sea brine pool microbial community for anticancer therapeutics

The hallmarks of cancer, which categorises the survival and proliferative mechanisms of cancer cells, was first described by Hanahan and Weinberg [1]. Although these aspects of cancer are well studied, and even with the advances in personalized medicine for patient tailored treatment, there still exists cancer forms for which limited treatment options are available. Breast cancer, the most prevalent cancer in women globally, has an estimated 1.67 million cases diagnosed in 2012 [2]. Women of Arab [3] and African descent [4] present breast cancer at an earlier age, have more aggressive tumour types with a high prevalence of triple negative breast cancer. Penta-negative tumours (i.e., negative for the estrogen and progesterone receptors, EGFR, HER2, and cytokeratin 5/6) have also been reported in Saudi women [5]. In our previous seminal investigations [6, 7], we reported the anticancer potential of extracts obtained from brine-pool microbes. Most of these extracts were active against estrogen receptor positive (ER+) breast cancer cells. Since the triple-negative type of breast cancer is a more aggressive form and as currently no therapies are available, we aimed to identify new sources of anticancer compounds that can pave the way to develop novel therapies for triple negative breast cancer.

Approximately 60 % of current anticancer therapeutics are derived from natural products, including, for example, marine-derived compounds such as cytarabine, trabectedin, eribulin, and dolastatins [8]. A review by Agrawal et al. [9] described nonribosomal peptides isolated from marine microbes having anticancer activity while in a research article Neelam et al. [10] discovered a marine halo-alkaliphilic bacteria species possessing anti breast cancer activity. This evidence supports the pursuit of mining marine environments for the discovery of new anticancer agents. Twenty-five (mostly) anaerobic deep-sea brine pools with extremely high salt concentrations have been reported in the Red Sea. Multi-extremophilic microbes that inhabit these environments are not only adapted to high salinity (4–26%), but also to elevated temperature, low oxygen concentrations, and high concentrations of heavy metals [11‐14]. These extreme marine environmental conditions favour the production of secondary metabolites and thus potentially unique and potent natural compounds. Extremophilic marine bacterial species from these environments, therefore, present a unique opportunity for discovering novel anticancer compounds [6, 7] that address the ever-changing need for improved chemotherapeutic drugs.

This study reports on anticancer activity of extracts from bacterial isolates from different habitats around brine pools within the Red Sea. We screened a total of 60 extracts against seven cell lines representing colorectal carcinoma, fibrosarcoma, breast carcinoma, cervical carcinoma, neuroblastoma, and normal cell lines. After initial screening, active extracts (> 30% growth inhibition) were selected for testing in selected cell lines to investigate if apoptotic activities were moderated by caspases (executors of apoptotic cell death).

As a follow-up from our previous studies [6, 7], we screened extracts from 60 marine bacteria isolated from brine pools of the Red Sea. We would like to emphasize that in our previous two investigations, we could only find extracts active against MCF-7 (ER+) cells, but the current study reports anticancer activities of the extracts isolated from the Red Sea brine-pool harboring bacteria against fibrosarcoma, cervical cancer and particularly BT20 (triple negative) cancer cells. Primary cytotoxicity screening of all extracts against seven cell lines representing five different cancers enabled us to broadly identify potential anticancer extracts from deep-sea microbes. All extracts were also screened against one normal fibroblast cell line (BJ) to further identify those who exhibited selectivity for cancer, but not normal cells.

We investigated whether the brine-pool location had any correlation with anticancer activity induced by the bacterial isolates. Of all isolates from the respective brine-pools, we found that about 50% of all isolates collected from Erba Deep (located at a depth of 2395 m), showed greater than 30% growth inhibition (Fig. 1). The most closely related validly described species to these strains are (Table 1): Chromohalobacter israelensis, Salinivibrio siamensis, Idiomarina seosinensis, Pseudoalteromona scarrageenovora, Pontibacillus halophilus, and Alteromonas macleodii. Our previous work [7] has reported anticancer activity of Chromohalobacter israelensis in HeLa cells. A PubMed search did not reveal even a single article that describes the anticancer activity of any of the other five bacterial species. This highlights the fact that the microbes found in the deep-sea brine pools of the Red Sea (especially Erba Deep) may have unique anticancer compounds that can be explored in the future to develop new drugs.

Apoptosis assays (APOPercentage and caspase-3/7 activity) confirmed that several of these selected (showing > 30% cell growth inhibition) extracts induced apoptotic cell death in cancer cell lines. This secondary screening process identified the extracts that selectively targeted a specific type of cancer via apoptotic cell death. We further investigated if extracts from a particular bacterial species have anticancer activity towards a specific cell line. Intriguingly, all three extracts (10, 15 and 22) that specifically inhibited the growth of fibrosarcoma cells (HT-1080) belonged to strains that were closely related to Salinivibrio costicola, Salinivibrio sharmensis, and Salinicola salaries, respectively, showing an enrichment of genus Salinivibrio. It would be interesting in future to investigate Salinivibrio extracts against other sarcomas as well. In MCF-7 cells, two out of four most active extracts (XI10 and 13) belonged to Pseudoalteromonas mariniglutinosa. This bacterial species had not been tested so far for anticancer activities (PubMed search). Secondary metabolites isolated from Psuedoalteromonas sp. off the coast of Brazil were reported to have potent anticancer activity against a leukemic and melanoma cell line, and the active compound, prodigiosin, was shown to be selective for cancers overexpressing ErbB-2 [19]. In BT20 cells, extracts (16 and 55) showed selective cell death, and these extracts belong to species Halomonas hamiltonii and Alcanivorax dieselolei, respectively, none of which have ever been shown to have anticancer activity before. Our observation that Halomonas sp. and Alteromonas sp. contributed to the majority of growth inhibition may be attributed to their ability to produce molecules including exopolysaccharides (EPSs) and Dithiolopyrrolone (DTP), respectively. Exopolysaccharides (heterogeneous polymers) isolated from Halomonas stenophila and Halomonas smyrnensis induced pro-apoptotic effects against human T-leukemia cells [20] and breast cancer MCF-7 cells [21]. Alteromonas sp. are also well known for producing dithiolopyrrolone (DTP) molecules, which are known potent natural antibiotics; DTP obtained FDA approval as topical antibiotic Bactroban® (GlaxoSmithKline) [22]. Not surprising, DTP also exhibits potent anticancer activity. These classes of antibiotic or polysaccharide type molecules most probably explain the anticancer activity we observed in this study.

In conclusion, our study has identified several microbial species that have the potential to kill selectively cancer cells, and interestingly many of these species have never been previously tested for their anticancer activities. Here, we provide seminal baseline data pinpointing which bacterial species and brine-pools should be targeted for future investigations to isolate anticancer compounds. This work is of particular importance for triple negative breast cancer therapeutic development as no drug exists till date that can effectively cure this aggressive form of breast cancer. Testing of these marine extracts against penta-negative breast cancer cells should be of great interest to future studies.